Quantitative analyses of various metabolities in body fluids, e.g., glucose, bilirubin, urea nitrogen, uric acid, cholesterol, lactate dehydrogenase, creatine kinase, GOT, GPT, etc., are of clinical significance and inevitable for the diagnosis, tracing of therapeutic progress, and prognostic judgement of disease. In clinical chemical examinations of body fluid samples, such as blood, it is desirable to make a precise analysis with a small amount of a liquid sample. A wet process using an analytical reagent in the form of a solution has hitherto been widely employed in this field, but it lacks the rapidity required for such analysis.
A dry chemical analysis using an analytical element in a substantially dry state is also known, in which a testing element or multi-layer analytical element having an analytical reagent system incorporated therein is used. The dry chemical analysis is superior to the wet chemical analysis in convenience, economy, and rapidness.
A dry multi-layer analytical element has been developed which allows a highly precise analysis to be conducted with a small amount of a liquid sample. Known dry multi-layer analytical elements include those disclosed in JP-B-53-21677 (the term "JP-B" as used herein means an "examined published Japanese patent application") and JP-A-164356 and 60-222769 (the term "JP-A" as used herein means as unexamined published Japanese patent application"). In one instance, a dry multi-layer analytical element is composed of a transparent support, a reagent layer, a reflective layer, and a developing layer. The transparent support is, for example, a thin plastic film which has been subjected to a subbing treatment. The reagent layer provided on the transparent support contains a reagent which reacts with a substance to be analyzed (analysis) to develop a color having an optical density corresponding to the amount of the substance under analysis. The reflective layer serves to prevent light transmitted through the reagent layer from reaching the spreading layer and to exclude the influence of a liquid sample remaining in the spreading layer on the optical measurement of the reagent layer. The spreading layer functions to spread a liquid sample dropwise added to an area substantially in proportion to the amount of the sample added. Quantitative analysis by the use of such a dry analytical element can be carried out by dropping a given amount of a liquid sample, e.g., whole blood, on the surface of the spreading layer, where the blood is spread over. The blood then passes through the reflective layer and reaches the reagent layer, where it reacts with a reagent to develop a color. After the dropwise addition of the sample, the analytical element is preserved at a constant temperature for an appropriate period of time to allow sufficient progress of the color development. Thereafter, light is irradiated on the element from the side of the transparent support, and the amount of the reflected light is measured at a specific wavelength to obtain a reflection density, from which the amount of the analysis can be obtained using a previously prepared calibration curve.
In either the wet and dry chemical analyses, it has been usual to use, as a sample, the serum or plasma prepared by separating red blood cells from whole blood. However, separation of red blood cells from the other blood components is laborious and requires cost by equipment. It is therefore desirable to make the analysis using undiluted whole blood.
In dry chemical analyses on whole blood, it is necessary to separate the blood cells, i.e., the red blood cells and white blood cells, and other high-molecular-weight components of the whole blood by a means provided within the analytical element. It has been proposed to separate the blood cells and high-molecular-weight components from the whole blood by means of a filter layer provided in an analytical element as disclosed in JP-B-53-21677. Nevertheless, as mentioned in JP-A-60-111960, it requires substantial time to remove the blood cells from serum or plasma by means of such a filter layer. Moreover, it is possible a part of the analysis may be lost during passage through the filter layer, causing errors in the analysis.
JP-A-60-279860 proposes a dry analytical element useful for analyzing a specific component in a whole blood sample, in which blood cells are separated and removed from the plasma within an analytical element and the component to be analyzed can be rapidly diffused into a reagent layer. This analytical element has a multi-layer structure comprising, in this order, a support, a first non-fibrous porous layer, a second non-fibrous porous layer, and a fibrous porous layer; the three layers being intimately bonded together in once piece with an adhesive locally applied between each adjacent surface so as not to substantially interfere with uniform penetration of a liquid sample therethrough. Any one of the three layers may contain a reagent composition which develops a color and the second non-fibrous porous layer has an average effective pore size of from 0.8 to 30 .mu.m.
However, when whole blood is analyzed with the multi-layer analytical element, the spectral absorption of the color developed by the reagent composition may be influenced by the amount of the sample added or diffusion in the developing layer. This is particularly so when the wavelength used for detection is close to the absorption wavelength of the hemoglobin in blood. Additionally, empirical results have shown that, with the same analyte content in the plasma, the analytical results vary depending on the hematocrit value (the volume percentage of blood cells in whole blood).